Given the increasing number of processes attributed to G proteins, some mechanism which directs articulation of individual receptors, G proteins and effectors is likely to exist. This might be particularly true in the nervous system, where rapid and discrete response is a hallmark of synaptic transmission. It has been observed that alpha subunits of G proteins may form complexes with synaptic membrane tubulin and undergo a directed transfer of nucleotide from the latter. This appears to be a highly specific process, as tubulin has been shown to bind, with high affinity, to only three G proteins, alpha s, alpha i1, and alpha q. Even though several other G proteins - alpha i2, alpha i3, alpha o and transducin (alpha r) - are quite closely related, their affinity for tubulin is much lower. The initial objective of research in this proposal is to determine the interactive domains on tubulin for alpha i1 or alpha s and those on the G proteins for tubulin. Chimeric G proteins which contain portions of high- and low-tubulin affinity G alphas and specific G protein mutants will be expressed and purified. These G proteins will be examined for their interaction with tubulin via AAGTP transfer (from tubulin) or stabilization of GTP bound to tubulin. Normal and mutant G proteins will also be fluorescently labeled and combined with fluorescently labeled tubulin so that kinetics of their interaction will be determined. These chimeric proteins as well as peptides corresponding to the tubulin-association regions on G proteins will be used to determine the physiologic relevance of tubulin activation of G protein. These studies will be done in vitro (with purified proteins), in permeable cells and in individual locus coeruleus neurons. Novel crosslinking agents will be synthesized and used to probe the articulating facets between tubulin and G protein as well as the nature of the GTP transfer process. These agents will also be used to determine the targets of tubulin in the cell. Reconstitution experiments will also be done to determine whether other constituents of the synaptic membrane which are known to interact with tubulin might influence the association or the transfer of nucleotide between tubulin and G proteins. Immunocytochemical studies will be done to localize tubulin-G protein complexes at the synapse and to determine whether prolonged neural activity might alter this distribution. It is hoped that these studies will provide insight as to how the cytoarchitecture of the neuron contributes to neuroreceptor response and responsiveness. Increase in the understanding of synaptic function will lead to increased understanding of the inner workings of the brain.